A critical role for miR-142 in alveolar epithelial lineage formation in mouse lung development

Abstract

The respiratory epithelium arises from alveolar epithelial progenitors which differentiate into alveolar epithelial type 1 (AT1) and type 2 (AT2) cells. AT2 cells are stem cells in the lung critical for the repair process after injury. Mechanisms regulating AT1 and AT2 cell maturation are poorly defined. We report that the activation of the glucocorticoid pathway in an in vitro alveolar epithelial lineage differentiation assay led to increased AT2 marker Sftpc and decreased miR-142 expression. Using miR-142 KO mice, we demonstrate an increase in the AT2/AT1 cell number ratio. Overexpression of miR-142 in alveolar progenitor cells in vivo led to the opposite effect. Examination of the KO lungs at E18.5 revealed enhanced expression of miR-142 targets Apc, Ep300 and Kras associated with increased β-catenin and p-Erk signaling. Silencing of miR-142 expression in lung explants grown in vitro triggers enhanced Sftpc expression as well as increased AT2/AT1 cell number ratio. Pharmacological inhibition of Ep300-β-catenin but not Erk in vitro prevented the increase in Sftpc expression triggered by loss of miR-142. These results suggest that the glucocorticoid-miR-142-Ep300-β-catenin signaling axis controls pneumocyte maturation.

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Acknowledgements

We acknowledge the precious help provided by Kerstin Goth and Jana Rostkovius in managing the mouse colonies, and Shirisha Bagari for the genotyping of the mice. C.M.C. and E.E.A. were funded by start-up grants from the Cardio Pulmonary Institute (CPI) (previously known as Excellence Cluster Cardio-Pulmonary System (ECCPS)). C.M.C. also acknowledges the support of the University Hospital Giessen and Marburg (UKGM). E.E.A. acknowledges the support of the University Hospital Giessen and Marburg (UKGM) and the German Center for Lung Research (DZL). S.B. was supported by grants from the Deutsche Forschungsgemeinschaft (DFG; BE4443/1-1, BE4443/4-1, BE4443/6-1, BE4443/14-1, KFO309 P7 and SFB1213-projects A02 and A04), UKGM, Universities of Giessen and Marburg Lung Center (UGMLC) and the Deutsche Zentrum fur Lungenforschung (DZL). J.S.Z was funded through a start-up package from Wenzhou Medical University and the National Natural Science Foundation of China (Grant Number 81472601). S.H. was supported by University Hospital Giessen and Marburg (FOKOOPV), the German Center for Lung Research (DZL), and Grants from the DFG (KFO309 P2/8; SFB1021 C05, SFB TR84 B2). CC was supported by the Interventional Pulmonary Key Laboratory of Zhejiang Province, the Interventional Pulmonology Key Laboratory of Wenzhou City, the Interventional Pulmonology Innovation Subject of Zhejiang Province, the National Nature Science Foundation of China (81570075, 81770074), Zhejiang Provincial Natural Science Foundation (LZ15H010001), Zhejiang Provincial Science Technology Department Foundation (2015103253) as well as the National Key Research and Development Program of China (2016YFC1304000). B.M. acknowledges the ANR-18-CE92-0009-01.

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Fig. S1:

Supplementary material 1 (JPEG 1410 kb) Nuclear and Cytoplasmic expression of miR-142 as well as FACS-based isolation of resident epithelial and mesenchymal cells and validation of gene expression. (A) Expression of miR142-3p and miR-142-5p in the nuclear and cytoplasmic compartment after cellular fractionation. (B) Efficiency of cell fractionation experiment shown by western blot analysis of nuclear and cytosolic fractions from the 3 weeks old adult WT lungs homogenate. (C) In situ hybridization on E18.5 lung sections for miR-142-3p and -5p with immunofluorescence for the AT1 marker Hopx. Please note the co-expression of miR-142-3p and -5p with the AT1 marker Hopx (yellow arrowhead: non specific signal; white arrow: specific signal showing cells co-expressing miR-142 and Hopx). (D,E) FACS-based isolation of resident epithelial and mesenchymal cells and validation of gene expression. (D) Experimental design for FACS-based isolation of CD31−ve CD45−ve Epcam+ve (resident epithelium) and Epcam−ve (resident mesenchyme). Quantification showing the conservation of the epithelial to mesenchymal ratio (around 1 to 3) at E14.5 and E18.5. (E) qPCR validation of the isolated epithelial and mesenchymal cells using epithelial (Cdh1, Epcam, Fgfr2b) and mesenchymal (Fgf10, Acta2, Vimentin) markers at these two time points

Fig. S2:

Supplementary material 2 (JPEG 895 kb) Combined loss of miR-142-3p and miR-142-5p invitro showed no branching defect during pseudoglandular stage of lung development (A) In vitro culture of E11.5 embryonic lung explants, treated with scramble, mo142-3p and mo142-(3p + 5p). (B) Corresponding qPCR analysis for the expression of miR-142-3p and -5p and their respective targets Apc and Ep300.Scale bar low mag: 100 µm, high mag: 25 µm

Fig. S3:

Supplementary material 3 (JPEG 709 kb) Gene Array and KEGG pathway analysis on E18.5 control and KO lungs. (A) Heat map of the most differentially expressed genes (according to their p values) between KO and Control lungs at E18.5 (n = 3). (B) Corresponding KEGG pathway analysis

Fig. S4:

Supplementary material 4 (JPEG 1016 kb) Quantification of the number of Epcam as well as AT1 and AT2 cells in Control and Experimental miR-142 KO lungs at E18.5. Analysis of proliferation (A) Flow cytometry analysis using Epcam and Sftpc antibodies in E18.5 miR-142 KO showing an increase in the percentage of AT2 cells compared to the wild type littermate controls. (B) Cdh1/Ki67 double IF staining showing no significant difference in proliferation in E18.5 Control and KO lungs. (C) qPCR analysis for general epithelial markers (Cdh1, Epcam) as well as for markers of the conducting (p63, Scgb1a1) and respiratory (Sftpc, Pdpn) airways in E18.5 Control and KO lungs. Scale bar D: low mag: 20 µm, high mag: 5 µm

Fig. S5:

Supplementary material 5 (JPEG 915 kb) Examination of Fgf signaling in E18.5 miR-142 Control and KO lungs. (A) qPCR analysis of Fgf10, Fgfr1b, Fgfr2b Fgfr2c, Sprouty2 and Etv5 in E18.5 Control and KO lungs. (B) Fgfr2 expression in the epithelium is validated by immunofluorescence staining with Fgfr2 and Cdh1 antibodies. Quantification indicating increased number of Fgfr2+ve cells in KO vs. control lungs. Scale bar B: low mag: 16 µm, high mag: 4 µm

Fig. S6:

Supplementary material 6 (DOCX 1037 kb) In vitro alveolar epithelial lineage formation. Flow cytometry analysis on the number of Epcam (A), bipotent (B), AT2 (C) and AT1 (D) cells in lungs cultured with morpholino miR-142-3p and -5p (mo-(3p +5p)) together. Expression of Sftpc and Pdpn upon IQ-1 treatment (E, F) or Erk inhibitor (G, H) treatment in E14.5 lungs grown in vitro with Scramble and morpholino specific to miR-142-3p and miR-142-5p. (I) Dose response curve for IQ1. Note that 2 µM IQ1, the lowest dose allowing Sftpc inhibition is still capable of blocking the effect of mo-5p on Sftpc expression

Supplementary material 7 (AI 1410 kb)

Fig. S7:

Supplementary material 8 (JPEG 192 kb) Expression of Sftpc in E14.5 Control and KO embryonic lungs grown 4 days in vitro (A) untreated conditions, (B) in presence of Erk inhibitor and (C) in presence of IQ1

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Shrestha, A., Carraro, G., Nottet, N. et al. A critical role for miR-142 in alveolar epithelial lineage formation in mouse lung development. Cell. Mol. Life Sci. 76, 2817–2832 (2019). https://doi.org/10.1007/s00018-019-03067-8

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Keywords

  • Alveolar epithelium
  • microRNA-142
  • Lung
  • EP300
  • Beta-catenin